The present invention relates to the preparation of albumin. More particularly, the present invention is directed to a method in which large-scale preparation of clinical albumin is facilitated. The method has the advantages of giving a significantly higher yield than prior art procedures and of requiring less than one-half the processing time of such known procedures as the Cohn procedure.
Previous large-scale procedures for the preparation of albumin have included those described by Cohn et al. in J. Amer. Chem. Soc. 68, 459-475 (1946) and J. Amer. Chem. Soc. 72, 465-474 (1950). Other procedures for the preparation of albumin have included that described by W. Schneider et al., Blut. 30: 121-134 (1975). Such prior art procedures, in general, have not resulted in satisfactory yields and have required long processing periods in order to prepare the albumin. The present invention for the preparation of albumin has the advantages of providing a significantly higher yield than the Cohn et al. procedure and of requiring less than one-half the processing time of the Cohn et al. procedure.
The present method for the preparation of albumin includes the steps of: (1) dilution of plasma in liquid form with an equal volume of an aqueous NaCl solution containing: (a) EDTA (diosodium ethylene dinitrilo tetraacetate) to break the iron-transferrin complex and possibly other metallo proteins; and (b) a stabilizer such as sodium caprylate: (2) heating the plasma-containing solution at approximately 60.degree. C. for about 11/2 hours at a pH of 6.2.+-.0.1; (3) cooling the solution to about 10.degree. C.; (4) precipitation of impurities of the heated solution with PEG 4000 (polyethylene glycol) at approximately 18-20% by weight concentration, leaving the albumin in the supernatant; (5) isoelectric precipitation of albumin from the PEG supernatant at a pH of about 4.6; and (6) recovering the resultant albumin which is devoid of the albumin dimer.
In the literature there is a description by Inman et al., Vox Sang. 6: 34-52 (1961) of a large-scale method for the purification of human transferrin. In the literature method, the use of EDTA and ethanol at low temperature is described for decreasing the solubility of transferrin during the purification of this protein. The method of the present invention includes the use of EDTA and heating, on the other hand, in order to reduce the stability of transferrin and other metallic proteins, thus effecting conformational changes which facilitate their precipitation by 20% PEG, leaving a highly purified albumin in the supernatant.
In an alternative method of the present invention, the steps include: (a) adjusting the pH of plasma in liquid form to about 6.7; (b) heating the plasma at a temperature of approximately 60.degree. C. for about 11/2 hours; (c) adjusting the pH of the plasma to about 5.7; (d) precipitating impurities from the plasma by the addition of ethanol in an amount sufficient to give a final concentration of about 40 to 44% by volume in the plasma, along with cooling of the plasma to about -5.degree. C., with the albumin remaining in the supernatant; and (e) adjusting the pH of the supernatant to about 4.8 to precipitate albumin from the supernatant.